Smooth muscle and nonmuscle myosin II are regulated by phosphorylation of the 20 kDa regulatory light chain (RLC) located in the neck region. This region contains a single alpha-helical segment of the myosin heavy chain and the RLC and the essential light chain (ELC). There are two vertebrate genes encoding for nonmuscle myosin IIs, termed IIA and IIB. Platelets express only the IIA isoform, whereas brain expressed predominantly the IIB isoform. Most other cell express nearly equal ratios of IIA and IIB where they are differentially localized. Previous work in our lab has revealed that nonmuscle myosin IIA and myosin IIB have differing enzymatic and motile properties. In order to better understand the basis for this difference, we have engineered myosin IIA and myosin IIB S-1 like fragments for baculviral expression. Preliminary characterization of the steady-state actin-activated MgATPase reveals that IIA S1 has a higher enzymatic rate than that of IIB S1. Both proteins express well. We are currently examining the transient kinetics of these two S1 isoforms. We have also expressed two mutant NMIIA HMM constructs corresponding to mutations found in patients with May-Hegglin Anomaly and Fechnter's Syndrome. These disorders are associated with giant platelets and/or deafness. We find that both the R702C mutation and the N93K mutation have profound effects on the enzymatic function, but the N93K mutation is almost entirely inactive. Finally we used transient kinetic techniques to show that the phosphorylation-dependent regulation of NMIIB requires two heads.